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Some terminology"Antigenicity": the ability to interact with an antibody"Immunogenicity": the ability to induce an immuneresponse
"Hapten": an antigen that is not immunogenic
HaptensImmunogens
Note : Immunogenicity does depend one os as we ...
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Epitopes
"Epitope" = "Antigenic determinant" =
- -
form a bimolecular complex
form a trimolecular complex
n so u e ant gen
no MHC involvement
Anti en can be rotein
una e to in so u e antigenMHC restricted
polysaccharide or lipid
Epitope is accessible, often Epitope is internal, denaturedconformational, hydrophilic
and mobile
linear peptide, hydrophobic and
MHC-bound
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Two kinds of B-cell epitopes
"Discontinuous" or "conformational" epitopes
" " " "
denature
denature
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Factors that influence immunogenicity
Part I : Contribution of the immunogen
Foreignness : a molecule must be recognized asnon-se .
Molecular size : 5 - 10 kDa is the minimum
Chemical composition and heterogeneity : themore complex, the better.
processing by APCs is an essential step.
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antibody Nopara
hapten
metacarrier binding
Weak
binding
conjugate
ort o
cross-react on
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Increase susce tibilit to ha oc tosis 1
Couple the antigen to beads
The protein can be covalently linked to commonly
available activated e. .CNBr-activated a arosebeads.
ternative y, re oo ce scan e use .Coupling agents include tannic acid, chromic chlorideor g utera e y e.
Anti ens can also be adsorbed to activated r norto colloidal goldparticles.
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Increase susceptibility to phagocytosis (2)
se e ec rop ore ca y pur e an genDirect injectionof a gel fragment (rabbits).
- Freeze the gel slize (liquid N2) and grind it in an eppendorf tube. Suspend in buffer.
Excise the blotted antigenfrom a nitrocellulose (NC) membrane- Finely grind the NC slice in a mortar and suspend in buffer, OR...
- Dissolve the NC slice (1-2 cm2) in 0.3 ml DMSO, slowly add to 3 ml 15 mMNaHCO3 bu er H 9.6 under vortexin . NC reci itates as ine artricles.
Sediment at 10,000g, wash twice with PBS and resuspend in 1 ml PBS, OR...- Directly implant the NC slice subcutaneously.
Use e ectro-e ut on o t e ant gen rom t e ge s ice. T e"trick" is to localize the target on the gel ...This can be achieved by:
- stainin a side stri- by using reversible gel staining methods (e.g.: Copper, Zinc, Nile Red, Sypro
Orange stain)
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Part II : Contribution of the host
Genot e of the reci ient animal : enetic control ofimmune responsiveness is largely confined to MHC
e use o a uvan s : reun s a uvan , a um e c.Immuno en dosa e and fre uenc : avoid both lowand high zone tolerance; use repeated injections.
oute o a ministration : i.v., i.m., s.c., i. ., i.p.etc.
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How do adjuvants work?
Antigen depot formation: a protected reservoir ofanti en or slow release to drainin l m h nodes.This hel s the
formation of memory cells and prolonged antibody responses.
Injection-site depots of antigen and adjuvant can induce theormat on o granu omas.
Delivery vehicle or inert carrier: help direct antigento perip era immune organs an serve to concentrate oraggregate the antigen for efficient delivery to the immunes stem. E. .: sur actants concentrate rotein molecules on alarge surface area.
of the immune system, resulting in cell proliferation and/ordifferentiation. Included are: mineral salts, surfactants, peptidesand cytokines.
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Ad uvants hel
Antibody t iters
virus alone virus and adjuvant
100% 100%0%
1 year
postvaccination postvaccination
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Adjuvants help
Postulated mode of action :
Freund's incom lete ad uvant
epo
formation
granu oma
formation
non-spec c
stimulation
+ + -
Freund's complete adjuvant (#)
Alum
+
+
+ +
+
+
-
Mycobacterium tuberculosis(*)Bordetella pertussis(**)
++--
--
y
Synthetic polynucleotides +
-
-
-
-
Active adjuvant components : (*) muramyl dipeptide (MDP);
(**) LPS and pertussis toxin (whooping cough); (***) lipid A
,
"Granuloma" : tumour-like mass, nodule of granulation tissue containing lymphocytes,
macrophages and fibroblasts; result of chronic inflammatory response.
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Some valuable alternatives to Freund's
Replace crude preparations (whole bacteria) by refined products.Refined immunostimulators include: Trehalose Dimycolate (TDM)
Monophosphoryl Lipid A (MPL), Muramyl Dipeptide (MDP), Lipoteichoid
Acids (LTA).
Replace mineral oil (paraffin) by metabolizable oils (squalene).Numerous commercial products are now available:
- Ribi Adjuvant System : TDM and or MPL, squalene
- TiterMax: co ol mer CRL-8941 s ualene
- Montanide ISA Adjuvants- Syntex Adjuvant Formulation (SAF): thr-MDP, squalene
- etc.
Cheap home-made alternatives:
- Aluminum Salt Adjuvants- Nitrocellulose-adsorbed Protein
- Entrapped antigen (acrylamide, liposomes = lipide bilayer vesicles )
- ISCOMS (immune-stimulating complexes = saponin/cholesterol
micelles)
- etc.
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'
... e sure to use syr nges
... be sure to thoroughly MIX Complete adjuvant
... make a "stiff mayonaise"
... in ect small volumes er site es eciall i.d.
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Immunization routes. . -
- no adjuvant, except for liposomes or iscoms
- no large particles
- anger o anap y ax s or so u ze an gens
- helps "spreading" the immune response over different
lymphoid tissues
I.d. - limited to small volumes
- severe hypersensitivity reactions may result in combination
'
S.c. - preferred route for booster
- slow Ag release minimizes the risk for anaphylaxis
- use e our corners o e an ma
I.p.: - best for large volumes
- ood access of the A to the l m hatics
I.m.: - preferred route for poultry
- relatively large volumes are possible
Other(incl. intrasplenic, intranodal, intrafootpad):
- only if the amount of available Ag is very limited.
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Immunogen dose : don't overshoot ...
There is a huge workable range.
Much depends on the immunogenicity of the molecule,
including foreignness and immunogen presentation.
Rules of thumb:- Mouse: 1 - 100 g
- Rabbit: 10 1000 g
- Chicken: 1 100 g
- Goat (Sheep): 20 - 2000 g
Use enough for the primary immunization but decrease the
dose for the boost injections!
- This helps selection towards antibodies with high affinity(affinity
" "
dose of antigen. Binding of its antigen is the first step in activation of
a memory cell.
-
contaminants start being produced. Thus: better specificity.
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A routine immunization schedule
Rabbit Mouse Goat
Dose u 50-1000 5-100 500-2000
Formulation
CFA - IFA (*)
CFA - IFA CFA - IFA
n ec on vo .Route
m 200 u 2 m
i.d. - s.c. s.c. i.d. - s.c.
Frequency
Sam le vol.
at least 3 weeks interval
20 ml 200 ul 200 ml
(*) 50-50 emulsion of adjuvant and antigen solution; first injection
with Com lete Freund's Ad the followin boosts with Incom lete
Freund's Adjuvant. ! SMALL volumes per site, esp. i.d.!!!!
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Troubleshooting ...
Antigen property Classificationof defect Result RemedyDefect
Lacks an epitope No Ag binding toB-cells
B-cell tolerance No response None
Lacks Class II
protein binding site
No Ag-fragmentpresentation
Non-responsiveness
No response orprimaryresponse only
Switch animalsor attach class IIbinding site
Lacks T-cell
recepter binding site
No - e per ce
involvement
-ce to erance No response or
primaryresponse only
w tc an ma s
or attach TCRbinding site
Nondegradable
Small sizeno T-helper cell
o g- ragmenpresentation
-responsiveness
Non-No response
primary resp.
Conjugate to
Nonparticulate
Form
Poor phagocytosis Weakerresponse
Self-polymerizeCouple to beads
-
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How about a leap into Phylogeny?
Use chickens...
million years): excellent for conserved antigens
on- nvas ve, easy an o y co ec on: eggs
Very productive: "1 chicken = 10 rabbits" (15% of
t e oo vo ume per 4 wee s) or 1 c ic en wee= 3 rabbit months"
Easy to keep
No interaction with rheumatoid factors, com lementsystem nor with Fc receptors
No bindin to rotein A or rotein G
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Serum can be used in crude form, egg yolk
nee s pur cat onStep 1: Separation of egg white and yolk- Freeze and thaw the eggs
- Roll the semi-solid yolk ball on absorbent paperStep 2: Delipidation of the yolk- Dilute yolk with 9 vol. of dH2O and mix thoroughly, pH to 7.0 with 0.1 M NaOH
- reeze e emu s on a -
- Spin down the lipids (20 min x 2,000-3,000g)- pH the supernatant to 7.6
Step 3: Purification of IgG (Y) by salting out
- Add 18.5 g powdered (NH4)2SO4 to 100 ml of delipidated yolk solutionunder constant stirring and leave stirring for 1 hour- Spin down IgG (20 min x 2,000-3,000g), dissolve in 10 ml TBS and
spin again. Collect the supernatant.- Repeat precipitation by addition of 1 vol. of 3.6 M (NH4)2SO4 if necessary
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Purification of chicken IgG made easy...
Promega "Eggstract"Separate yolk from egg
white using EggSeparator
"A 60 (75) minute protocol yields
PrecipitateLipids
- mg o pure g
per egg."
A PrecipitationSolution A, mix,
centrifuge
Add PrecipitationSolution B, mix,
centrifuge
PrecipitateIgY
Solubilize IgY to desiredconcentration
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Some applications need minor adaption
Immune complexes with chicken IgG are moresoluble. Gel diffusion results can be improved by addition
of PEG 6000 (2-6%) or 1.5M NaCl.Chicken IgG has a lower isoelectric point (pI) thanmammalian IgGs.
E.g.: rocket electrophoresis needs to be run at pH 5.5-6,not at pH 8.6.
Pepsin does not work for digestion into (Fab)'2 (but
is enerally not needed either).Chicken IgG is more susceptible to aggregation onfreezin and thawin . Stora e at 4? C is re erable.
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Immunization of chickens
Emulsify 0.5 ml of antigen solution (1 to 100 g)and 0.55 ml of Complete Freund's Adjuvant or RibiAdjuvant System.
In ect 0.5 ml of the emulsion into each breastmuscle.
'
Adjuvant or Ribi.
'pointless, can yield severe hypersensitivity reactions
and ma result in cessation of e roduction ! .
A fast and strong antibody response is routinely.
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